The publications and other materials used herein to illuminate the background of the invention, and in particular, cases to provide additional details respecting the practice, are incorporated by reference.
Drug and population screenings have become an essential part of the studies and diagnosis towards improved medical treatment and health care. Within the past two decades, biotechnology has transformed the pharmaceutical industry by providing detailed insights into disease processes and increasing number of drug targets. High-throughput screening technologies (HTS) for large compound libraries have emerged which has resulted in promising early-stage drug candidates. In theory, the wealth of new disease data and the abundance of early-stage assay hits from HTS programs should result in more drugs reaching the market. In practice, however, the pharmaceutical industry is failing to turn potential into profit, and only a small number of promising compounds will make it successfully through development. In order to be competitive, e.g. in finding leads in target molecule screening, rapid, efficient, cost-effective and simple screening techniques are required. A number of separation-free methods has been developed to meet the demands of ultra high-throughput screening in drug discovery: scintillation proximity assay (SPA, Amersham Biotech), amplified luminescence proximity homogeneous assay (ALPHA, Perkin Elmer Life Science), homogeneous time-resolved fluorescence assay (HTRF, Perkin Elmer Life Science and Schering), lanthanide fluorescence energy transfer assay (LANCE, Perkin Elmer Life Science), fluorescence polarization and correlation assays. The impressive list of various technologies includes methods based on radioactive scintillation, fluorescence polarization or correlation and time-resolved fluorescence. Other separation-free methods are also recognized and used in detection of sample substances in a wash-free assay set-up. Two-photon excitation, fluorescence cross-correlation and flow cytometric assay systems are examples of such methods.
The methodologies rely on the use of specific binding substances such as antibodies which recognize the substance of interest with high specificity and affinity. The methods can be used in competitive assay format or cleavage assays where substance is cleaved to separate detection elements and to generate signal. Sandwich type assay systems are often difficult to perform in resonance energy transfer technologies (RET) due to the fact that the detection principle is distance dependent. The distance, in a sandwich type assays, is typically too long to keep donor and acceptor elements close enough to generate adequate signal. Moreover, many of the technologies require labeling of different binding partners with different labeling agents. This may be extremely cumbersome and expensive.
In order to reduce background problems related to many assay technologies, a number of techniques has been developed. In RET assays, reduced or non-existing spectral overlap of donor and acceptor has been proposed (U.S. Pat. No. 6,245,514, U.S. Pat. No. 5,998,146). An efficient way of reducing background signal in an assay system is the use of highly specific and high affinity labeled capture probes against non-bound reaction substances. First specific binding partners are allowed to react and, thereafter, masking is performed using highly specific and high affinity capture quenching elements against non-bound binding partners (US 2004/0253593, U.S. Pat. No. 4,256,834, U.S. Pat. No. 4,404,366). The affinity of a high affinity interaction is thought to be above 1×107 M−1 and below 1×107 M−1 for low affinity interaction (Journal of Endocrinology, 2002, 175, 121). There are number of optical reduction methods which rely on particle carriers. In these methods, a specific signal is expressed on the particles. Detection is then carried out in a small volume containing the particle(s). Therefore, the background signal can be reduced drastically by sorting particles into the detection volume and carefully selecting the size of the detection volume (U.S. Pat. No. 6,177,277, U.S. Pat. No. 5,981,180, Clinical Chemistry 1997:43; 1749-56, Analytical Chemistry 2000:72; 5618-24, Analytical Biochemistry 1999:271; 143-51). A surface and optical reduction method in combination with dyes has also been investigated. In this method, a non-bound labeled binding partner is quenched using nonspecific dyes. A specific signal is expressed on an immobile, typically essentially flat, solid surface and a quenching component is used to reduce the signal in the solution starting from the vicinity of the surface toward the solution. The quenching component is used to limit the light penetration in the solution. The quenching molecules are used to narrow the thickness of the light penetration depth as excitation light is directed through the immobile solid surface into the solution and the signal towards the excitation light is monitored through the immobile solid surface. The method requires a immobile solid surface and very specific detection geometry in combination with a soluble dye molecule in solution. The method is performed in a separation-free assay format on a solid phase. Winkler has studied protein interaction of fluorochromes and used solution-based quenching mechanism to obtain separation-free fluorescence detection (Biochemistry, 1969:8; 2586). The investigation has been performed for studying interaction of a fluorochrome and proteins. The methodology used is not suitable for assay purposes. Jenkins et al have shown how DNA ruthenium intercalating compounds can be nonspecifically attached to DNA (Biochemistry 1992:31; 10809). Non-attached intercalating compounds are quenched in solution using Fe(CN)64−. Intercalation takes place through nonspecific interactions after nucleic acid hybridization. None of the nucleic acid strands are being labeled with the intercalating compound and, therefore, the intercalation compound is considered a nonspecific binder without prior coupling to a carrier molecule—in this case coupling to one of the nucleic acid strands.
Many of the abovementioned methods such as ALPHA, HTRF, LANCE and fluorescence correlation assays are suitable for small molecule assay formats but the methods are very cumbersome or impossible to develop for whole cell assaying. The distance dependence or focal restrictions limit the use of the methodologies. Fluorescence polarization can be used for whole cell assaying of typically above 1 nM concentrations of sample substance.